Mismatch Repair (MMR) of errors during DNA replication is essential for human life. Deficiencies in MMR cause a mutator phenotype and is the underlying cause of hereditary non-polyposis colorectal cancer. Helicase activity facilitates mismatch removal and is required for bacterial MMR. Our preliminary results show that certain members of the human RecQ family of DNA helicases dramatically stimulate DNA excision in a mismatch-specific manner. This suggests that human DNA helicases participate in MMR in ways that have not been previously appreciated. The present proposal will test the hypothesis that mammalian helicases play an essential role in MMR by regulating the mismatch removal step. In this proposal, we describe a systematic approach to define the role of human RecQ helicases in MMR. Using our in vitro reconstitution system we will explore the mechanisms of MMR stimulation. Furthermore, we will extend the studies to cell-based experimental systems through genetic experiments, confocal microscopy, and functional tests. Aim 1 is to determine physical and functional interactions between RecQ helicases and MMR proteins and their role in MMR. Aim 2 is to study role of RecQ helicases in MMR through genetic complementation and cell-based analyses. Unveiling the biological role of RecQ helicases in mismatch repair will not only open a new level of mechanistic understanding of the human MMR pathway and its involvement in cellular response to environmental toxins such as chromium, it may also lead to development of new diagnostic markers and more effective therapeutic strategies for treating cancer and other diseases associated with MMR deficiency.